Project Summary/Abstract A fundamental question in cell biology is how targeted intracellular protein trafficking is achieved and regulated. An excellent framework to ask this question is to study transport to a specific organelle or intracellular compartment. Trafficking of proteins to and into the eukaryotic flagellum is an ideal model to study polarized transport given that flagellar protein synthesis and trafficking can be induced experimentally on-demand, cargo proteins have been identified through proteomics and the ultimate cargo destination is localized to a very small region at the apical cell surface. This trafficking pathway was previously thought to only require microtubules and the regulation of microtubule motors through signaling pathways. Through quantitative analysis of flagellar motor dynamics in the canonical flagellar model system Chlamydomonas reinhardtii, we discovered that actin and an actin-based myosin motor play an important role in regulating the localization and compartmentalization of flagellar proteins. We also identified a variety of signaling pathways including a phosphatase, MKP-2, that are required for proper flagellar assembly. The broad goals of our work are to: 1) use chemical and genetic screening to identify novel pathways that integrate to control flagellar protein trafficking and molecular motors flagellar entry; and 2) use a toolbox of cellular and molecular assays to dissect the mechanisms by which they exert this control. We expect to uncover entirely new avenues for the study of secretory pathways conserved in all eukaryotes as well as novel functions for known genes in coordinated cellular trafficking.